Pancreatic adenocarcinoma is one of the most deadly malignancies in human for which there are very limited treatment options. There is great interest in the possible role of embryonic signaling pathways in the initiation and progression of this cancer and others. Among these pathways is canonical Wnt signaling, which is involved in the proliferation and self-renewal of stem/progenitor cells and has been speculated to have a similar role in cancer stem cells. The presence of activated beta-catenin in pancreatic tumor samples suggests that 30-40% of these tumors manifest Wnt signaling. Moreover, Matthias Hebrok and colleagues at UCSF have discovered that active Wnt signaling is required for the growth of several pancreatic adenocarcinoma cell lines in vitro. Our exploratory R21 project proposes to investigate the role of two novel sulfatases, called HSulf-1 and HSulf-2, in Wnt signaling within pancreatic adenocarcinomas and their contributions to the growth and tumorigenicity of these cancer cells. The Sulfs are extracellular enzymes which act on heparan sulfate proteoglycans (HSPGs) to remove a specific sulfation modification (glucosamine-6-O-sulfation). One known biological activity of the Sulfs is to modulate the interaction of Wnt ligands with HSPGs and to potentiate the ability of the Wnts to activate their signal transduction receptors. Our preliminary studies have found the expression of SULF1 or SULF2 transcripts in 23 of 24 pancreatic adenocarcinoma cell lines and the presence of Sulf-1 or Sulf-2 protein in 4/4 of these cell lines and 3/5 of pancreatic tumor samples. Expression of a dominant negative form (enzymatically-inactive mutant) of either Sulf-1 or Sulf-2 in 3 out of 4 of these lines resulted a reduction in Wnt signaling. Furthermore, co-culturing the same three cell lines in the presence of inactive Sulf protein produced a parallel reduction in Wnt signaling and cell growth in vitro. The proposed research will employ lentivirus-transduced shRNA expression to silence one or both Sulfs in representative pancreatic adenocarcinoma cell lines. Our Aims are: 1) To determine the effects of Sulf silencing on the growth and Wnt signaling of these cells in vitro; and 2) To determine the effects of Sulf silencing on the ability of the cells to form tumors in nude mice. Positive results from these studies should stimulate considerable interest in the Sulfs as possible targets (for small molecules or function-blocking antibodies) for the treatment of pancreatic adenocarcinoma in humans. [unreadable] [unreadable] [unreadable]